Inducible gene expression composition for using eukaryotic pol-2 promoter-driven transcription in prokaryotes and the applications thereof

ABSTRACT

Eukaryotic protein-coding messenger RNAs and non-coding microRNAs are naturally transcribed by type II RNA polymerases (pol-2) but not prokaryotic RNA polymerases. As a result, current eukaryotic RNA and protein production is performed either using eukaryotic pol-2 promoters in hybridomas or mammalian cells or using prokaryotic promoters in bacterial cells. However, because prokaryotic RNA transcription tends to be error-prone, frequent mutation is a big problem. Also, growing hybridomas or mammalian cells is relatively laborious and costly. To overcome these problems, the present invention provides a novel inducible composition and method for producing eukaryotic RNAs and/or their related peptides/proteins directly using eukaryotic pol-2 promoter-driven gene expression in fast growing bacteria, without the need of changing to prokaryotic promoters or growing hybridomas/mammalian cells. The RNAs and peptides/proteins so obtained can be used to develop drugs, cure diseases, treat tumors/cancers, produce pluripotent stem (iPS) cells, enhance wound healing, and make foods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of copending application Ser. No.13/572,263, filed on Aug. 10, 2012, which claims the benefit under 35U.S.C. §119(e) to U.S. Provisional Application No. 61/522,843, filed onAug. 12, 2011, all of which are hereby expressly incorporated byreference into the present application.

BACKGROUND OF THE INVENTION

Field of Invention

This invention generally relates to a composition and its applicationfor producing ribonucleic acids (RNAs, i.e. messenger RNAs andmicroRNAs) and/or proteins/peptides (i.e. antibodies and enzymes) usingeukaryotic RNA promoter-driven transcription in prokaryotes.Particularly, the present invention teaches a composition and its usefor generating RNAs and/or proteins/peptides using eukaryotic type IIRNA polymerase (pol-2) promoter-driven transcription in bacterial cells.Alternatively, the present invention is also an inducible geneexpression composition using chemical agents rather than antibiotics tostimulate eukaryotic RNA promoter-driven transcription in prokaryotes.The novelty of the present invention is to induce a quick adaptation ofprokaryotic cells to use eukaryotic pol-2 promoters for directlyexpressing desired RNAs and/or proteins/peptides without the need ofchanging to error-prone prokaryotic promoters or growing laborious andcostly hybridomas or mammalian cells and to improve the reading fidelityof the transcription in the prokaryotes. The ribonucleic acids andproteins/peptides so obtained can be used to develop drugs, curediseases, treat tumors/cancers, produce pluripotent stem (iPS) cells,enhance wound healing, and provide food supply.

Description of Related Art

As learning from current textbooks, any one of ordinary skill in the arthas known very well that prokaryotic and eukaryotic transcriptionmachineries contain differences and are not compatible to each other.For example, based on current understandings, eukaryotic RNA polymerasesdo not bind directly to promoter sequences and require additionalaccessory proteins to initiate transcription, whereas prokaryotic RNApolymerases form a holoenzyme that binds directly to promoter sequencesto initiate transcription. It is also a common knowledge for an ordinaryskill in the art to know that eukaryotic messenger RNA (mRNA) issynthesized in the nucleus by type II RNA polymerases (pol-2) and thenprocessed and exported to the cytoplasm for protein synthesis, whereasprokaryotic RNA transcription and protein translation take placesimultaneously off the same piece of DNA in the same place. Prokaryotessuch as bacteria and archaea do not have a nucleus-like structure. Thesedifferences make a prokaryotic cell difficult or even impossible toproduce eukaryotic RNAs and peptides/proteins using eukaryotic RNApromoters.

Prior art attempts at producing mammalian peptides and/or proteins inbacterial cells, such as U.S. Pat. No. 7,959,926 to Buechler and U.S.Pat. No. 7,968,311 to Mehta, used bacterial or bacteriophage promoters.For expression, the complementary DNA (cDNA) of a desired gene wascloned into a plasmid vector behind a bacterial or bacteriophagepromoter. The cDNA of the desired gene must not contain any non-codingintron because bacteria do not have RNA splicing machineries to processthe intron. Then, the vector so obtained was introduced into a competentstrain of bacteria, such as Escherichia coli (E. coli), for expressingthe desired gene transcripts (mRNAs) and further translating the mRNAsinto proteins. Nevertheless, these bacterial and bacteriophagepromoters, such as Tac, Lac, T3, T7, and SP6 RNA promoters, are notpol-2 promoters and their transcription is an error-prone process thattends to cause mutations. On the other hand, Mehta also taught thatglycerol might be used to increase the efficiency of bacterialtransformation; however, no description was related to enhancingpromoter-driven RNA transcription, in particular pol-2 promoter-driventranscription. Due to lack of possible compatibility between eukaryoticand prokaryotic transcription systems, these prior arts were stilllimited by the use of prokaryotic RNA promoters in prokaryotes.

Traditional inducible gene expression methods, such as the old teachingfrom Gossen M. and Bujard H. (1992) and U.S. Pat. No. 5,464,758 toGossen, required the use of antibiotics (e.g. tetracycline ordoxycycline) to stimulate the activation and expression of atetracycline-responsive-element (TRE)-controlled cytomegaloviral (CMV)or pol-3 (U6) promoter, namely Tet-On promoter. However, these Tet-Onpromoters are not eukaryotic pol-2 promoters and have never been testedin prokaryotes. Hence, if we can induce the adaptation of prokaryotictranscription machineries to express from eukaryotic pol-2 promoters, anovel inducible gene expression system will be made simply based on thedifferences between prokaryotic and eukaryotic transcription mechanismsrather than the previously toxic induction methods of using antibiotics,which may inhibit the growth of prokaryotes.

SUMMARY

The present invention provides a novel breakthrough to the old textbookconcept regarding incompatibility between prokaryotic and eukaryotictranscription systems. By adding some chemical agents, we now can inducea quick adaptation of prokaryotes for using eukaryotic pol-2 promotersto produce desired RNAs and the related peptide/protein thereof.

An object of the present invention is to provide an inducible geneexpression composition for using eukaryotic pol-2 promoter-driventranscription in prokaryotes and the applications thereof.

Additional features and advantages of the invention will be set forth inthe descriptions that follow and in part will be apparent from thedescription, or may be learned by practice of the invention. Theobjectives and other advantages of the invention will be realized andattained by the structure particularly pointed out in the writtendescription and claims thereof as well as the appended drawings.

To achieve these and/or other objects, as embodied and broadlydescribed, the present invention provides a composition useful forregulating eukaryotic promoter-driven gene expression in prokaryotes,comprising a chemical agent, containing a structure similar to3-morpholinopropane-1-sulfonic acid (MOPS), ethanol, glycerin, or amixture thereof.

In another aspect, the present invention provides a composition forregulating eukaryotic promoter-driven gene expression in prokaryotes,comprising: (a) at least a chemical agent capable of inducing orenhancing eukaryotic promoter-driven gene expression, wherein saidchemical agent containing a structure similar to3-morpholinopropane-1-sulfonic acid (MOPS), ethanol, glycerin, or amixture thereof; and (b) a plurality of prokaryotic cells, saidplurality of prokaryotic cells containing at least a gene mediated by aeukaryotic promoter-driven expression mechanism; wherein (a) and (b) aremixed together under a condition to induce the eukaryoticpromoter-driven gene expression of said gene in said prokaryotic cells.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory and areintended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A and 1B show an inducible pol-2 promoter-driven gene expressioncomposition (A) and its mechanisms (B) for RNA transcript and proteinproduction in prokaryotes and eukaryotes and microRNA (miRNA) productionin eukaryotes.

FIG. 2 depicts the results of bacterial culture broths treated with(left) or without (right) the mixture of 0.1% (v/v) MOPS and 0.05% (v/v)glycerin.

FIG. 3 shows the results of different bacterial pellets after treatedwith 0.1% (v/v) MOPS.

FIG. 4 shows the inducibility of different chemicals for inducing pol-2promoter-driven gene expression in competent E. coli DH5alpha cells.

FIG. 5 shows the Western blotting results of red RGFP protein expressioninduced by MOPS, glycerin and ethanol, respectively.

FIG. 6 shows the Northern blotting results of miR-302 and its pre-miRNAcluster expression induced by MOPS, glycerin and ethanol, respectively.

FIG. 7 shows iPS cell generation using miR-302 and/or pre-miR-302isolated from bacterial extracts (BE), which is confirmed by Northernblot analysis as shown in FIG. 6.

FIG. 8 shows the global DNA demethylation of Oct4 and Sox2 genepromoters induced by the miR-302 and/or pre-miR-302 isolated frombacterial extracts (BE).

FIGS. 9A-9C and 10A-10B show in vitro tumorigenicity assays of varioustumor/cancer cells in response to miR-302 and/or pre-miR-302 treatment.

FIGS. 11A-11C show in vivo tumorigenicity assays of embryonalteratocarcinoma Tera-2 cells in response to either the whole miR-302familial cluster (Tera2+mir-302s) or antisense miR-302d only(Tera2+mir-302d*) treatment (n=3, p<0.05).

FIG. 12 shows the results of an in-vivo wound healing trial usingmicroRNA miR-302 and/or pre-miR-302 containing ointment to treat skinopen wounds in mice.

DETAILED DESCRIPTION

The principle of the present invention is relied on the different andincompatible properties between prokaryotic and eukaryotic genetranscription systems. Normally, prokaryotic RNA polymerases do notrecognize eukaryotic promoters and vise versa. However, the presentinvention has identified a plurality of chemical agents that can serveas transcription inducers to induce and/or enhance the eukaryoticpromoter-driven gene expression in prokaryotes. The same or otherchemical agents may also serve as transcription inducers to induceand/or enhance the prokaryotic promoter-driven gene expression ineukaryotes. Hence, the novelty and knowledge taught in the presentinvention has provided a complete breakthrough to current understandingin the differences between prokaryotic and eukaryotic transcriptionmechanisms.

The transcription inducers found in the present invention are normallynot preferred to be used in a cell culture condition. In all chemicalstested, the top three most potent inducers are3-morpholinopropane-1-sulfonic acid (or named3-(N-morpholino)propanesulfonic acid; MOPS), glycerin and ethanol. Theirinduction capability is found to be dose-dependent in proportional totheir concentrations. Some chemicals containing a structure similar toMOPS, ethanol and/or glycerin may also have the same functionality. MOPSis frequently used as a buffering agent in bacterial lysis and plasmidextraction and hence is not recommended for bacterial cell culture.Ethanol is known to be a bacterial sanitizer. Glycerin can be used toincrease transformation efficiency by destabilizing bacterial cellwalls, yet is not recommended for bacterial cell culture. Both glycerinand ethanol are also viable preserving agents in that glycerin isbacteriostatic and ethanol is bactericidal in its action, respectively.In view of all these known functionalities to MOPS, ethanol andglycerin, an ordinary skill in the art would not anticipate the use of atrace amount (0.01% to 1% volume/volume concentration) of thesechemicals for inducing eukaryotic promoter-driven gene expression inprokaryotic cells without first knowing the knowledge of the presentinvention.

The present invention is an inducible gene expression composition usingcertain chemical agents to stimulate and/or increase eukaryoticpromoter-driven RNA transcription and the related peptide/proteinsynthesis in prokaryotes. An inducible gene expression compositioncomprises (a) at least a chemical agent containing a structure similarto MOPS, ethanol or glycerin, or a mixture thereof; and (b) a pluralityof prokaryotic cells that contain at least a gene mediated by aeukaryotic pol-2 promoter-driven or a pol-2 compatible viralpromoter-driven expression mechanism; wherein (a) and (b) are mixedtogether under a condition to induce the expression of said gene. Thepresent invention also provides a novel composition design and itsapplication for inducing a quick adaptation of prokaryotes to useeukaryotic pol-2 promoters for directly expressing RNAs and/orproteins/peptides of interest without the need of changing toerror-prone prokaryotic promoters or growing laborious and costlyhybridomas or mammalian cells and for improving the reading fidelity ofprokaryotic transcription, i.e. providing a pol2-like transcriptionmechanism. The RNAs expressed are preferably eukaryotic RNAs (i.e.messenger RNAs and microRNAs), and the proteins/peptides includesantibodies and enzymes.

Preferably, said prokaryote is a bacterial cells in particular,Escherichia coli (E. coli), and said chemical agent is3-morpholinopropane-1-sulfonic acid (MOPS), ethanol or glycerin, or amixture thereof. Also preferably, said eukaryotic RNA promoter is eithera eukaryotic pol-2 promoter (i.e. EF1alpha) or a pol-2 compatible viralpromoter (i.e. cytomegaloviral promoter or retroviral long terminalrepeat promoter). The gene mediated by said eukaryotic RNA promoter iscoded for either a non-coding or a protein-coding RNA transcript, orboth, selected from the group consisted of microRNA (miRNA), smallhairpin RNA (shRNA), small interfering RNA (siRNA), messenger RNA(mRNA), their precursors and homologs, and a combination thereof. Thepeptide/protein generated by the present invention is translated fromthe above protein-coding mRNA transcripts and may be selected from, butnot limited to, the group consisted of enzyme, growth factor, antibody,insulin, botulinum toxin (botox), any functional protein and ithomologs, and a combination thereof. Preferably, said condition forinducing the expression of said gene is a bacterial culturing conditionin Luria-Bertani (LB) broth at 37° C. with the addition of said chemicalagents.

To demonstrate the inducibility of said chemical inducers for RNA andprotein production in prokaryotes, the present invention adopted andmodified a lentiviral plasmid vector, e.g. pSpRNAi-RGFP-miR302, from theU.S. patent application Ser. No. 12/149,725 and Ser. No. 12/318,806 toLin, and then used the red fluorescent protein (RGFP) gene cloned intothe vector as a visible marker for measuring the process of RNAtranscription and protein synthesis, as shown in FIG. 1B. The originalcytomegaloviral (CMV) promoter of the pSpRNAi-RGFP-miR302 vector hasbeen replaced by human pol-2 EF1alpha promoter in this invention andherein formed a new plasmid vector, named pLenti-EF1alpha-RGFP-miR302,as shown in FIG. 1A. Additionally, since prokaryotes can not process anyin-frame intron, the SpRNAi intron of the RGFP gene has been removed andthe original intron-encoded miR-302 cluster was moved to the5′-untranslated region (5′-UTR) of the RGFP gene, so as to form aRGFP-miR302 gene. Broadly speaking, the 5′-UTR and 3′-UTR of a gene canbe considered as an extension of intron. Thus, miR-302 expressiondisclosed here is still based on the same intronic microRNA biogenesismechanism described in the scope of prior U.S. patent application Ser.No. 12/149,725 and Ser. No. 12/318,806. Advantageously, due to lack ofRNA splicing in prokaryotes, the miR-302 transcripts so obtained in thepresent invention remain as hairpin-like microRNA precursors (pre-miRNAor pri-miRNA), which can be further purified and delivered intoeukaryotic cells for forming mature miR-302 and eliciting the functionof miR-302 (FIG. 1B).

Induction of Eukaryotic Promoter-Driven Protein-Coding Gene Expressionin Prokaryotes

Escherichia coli (E. coli) is transformed by thepLenti-EF1alpha-RGFP-miR302 plasmid vector using, a z-competent E. colitransformation kit (Zymo Research, Irvine, Calif.) and cultivated inLuria-Bertani (LB) broth at 37° C. with frequent agitation at 170 rpm.After overnight incubation, the E. coli culture supplemented with amixture of 0.1% (v/v) MOPS and 0.05% (v/v) glycerin expresses highlyabundant red RGFP proteins that clearly stain the bacterial LB broth inred, whereas the blank control culture fails to produce any RGFP, asshown in FIG. 2. The presence of functional RGFP indicates that both itsRNA and protein are successfully produced.

To further confirm the specificity of protein induction by theidentified chemicals like MOPS, two transformed E. coli strains areprepared: one carries a pLVX-Grn-miR302+367 plasmid vector having a CMVpromoter-driven green fluorescent protein (GFP) gene and the othercarries the aforementioned pLenti-EF1alpha-RGFP-miR302 vector. Afterovernight incubation with 0.1% (v/v) MOPS, the E. coli transformed withpLVX-Grn-miR302+367 produces green cells while the other withpLenti-EF1alpha-RGFP-miR302 shows red cells, as shown in FIG. 3. Thisresult indicates that chemicals like MOPS can induce specific RNA and itrelated protein production either from a eukaryotic pol-2 promoter or apol-2 compatible viral promoter. Also, the induced RNA and proteinproduction is highly specific and can be regulated by the chemicaladded. It is particularly noted that the protein production is soabundant that even the bacterial cells are visually stained byrespective colors.

Among all chemicals tested in the present invention, the top three mostpotent inducers are MOPS, glycerin and ethanol, as shown in FIG. 4. Thequantitative result of the induced RGFP protein production is furtherconfirmed by Western blot analysis, as shown in FIG. 5 and Example 3.Bacterial RuvB protein is served as a house-keeping standard tonormalize the RGFP expression. The inducibility of these identifiedinducers is also found to be dose-dependent in proportional to theirconcentrations. Without any treatment, negative control E. coli cellsjust show their original bacterial color in absence of any fluorescentstain. Therefore, according to all these results, the present inventionclearly provides a novel chemical-inducible composition and itsapplication for modulating eukaryotic pol-2 promoter-driven or pol-2compatible viral promoter-driven gene expression in prokaryotic cells.The gene product can be either RNA or protein/peptide, or both. In viewof the above demonstration, it is very obvious for an ordinary skill inthe art to use other intron-free genes or the related cDNAs in place ofthe RGFP gene for producing functional RNAs and/or proteins inprokaryotes.

Induction of Eukaryotic Promoter-Driven Non-Coding RNA Expression inProkaryotes

As mentioned above that the pLenti-EF1alpha-RGFP-miR302 vector containsa microRNA miR-302 cluster in the 5′-UTR of the RGFP gene (FIGS. 1A and1B), the induced expression of the RGFP gene will also generate themiR-302 cluster (pre-miR-302s) as shown in the schematic mechanism ofFIG. 1B. Due to lack of RNA splicing machinery (e.g. spliceosome) inprokaryotes, miR-302 cluster so obtained in the present invention arefound to remain as hairpin-like microRNA precursors (pre-miR-302s orpri-miR-302s), which are very useful for being isolated and deliveredinto eukaryotic cells. In eukaryotic cells, these pre-miR-302s orpri-miR-302s can be further processed into mature miR-302s (i.e. themiR-302 microRNAs) for eliciting the miR-302 function. Similarly, otherkinds of microRNAs and the related precursors thereof can be producedfollowing the same protocol for miR-302 expression. Alternatively, somenon-coding RNAs such as short interfering RNAs (siRNAs) and smallhairpin RNAs (shRNAs) can be designed to mimic the above microRNAexpression. These non-coding RNAs preferably contain at least a sequencesharing 30% to 100% homology to a microRNA. Also, these shRNAs/siRNAsmay contain perfectly matched hairpin stem regions, while mammalianmicroRNA precursors (pre-miRNAs or pri-miRNAs) often contain mismatchedbase pairs. Given that most of microRNAs function as specific genesilencers and may play a variety of distinctive roles in manyphysiological and pathological mechanisms, including but not limited tobiological development, stem cell generation, nuclear reprogramming,cell differentiation, cell cycle regulation, tumor suppression,immunological defense, apoptosis, rejuvenation, wound healing, and manymore, the potential applications in theses pharmaceutical andtherapeutical fields are highly expected. Both of the transduced plasmidvector and the non-coding RNA (i.e. microRNA/shRNA) can besimultaneously amplified in the prokaryotic cells, such as E. coli. Themethod for isolating the amplified pLenti-EF1alpha-RGFP-miR302 plasmidDNA and the transcribed pre-miR-302s/pri-miR-302s is disclosed inExamples 5 and 6. The method for delivering the amplified non-coding RNA(i.e. pre-miR-302s/pri-miR-302s) and/or the vector (i.e.pLenti-EF1alpha-RGFP-miR302) into eukaryotes may be selected from thegroup of endocytosis, glycerol infusion,peptide/liposomal/chemical-mediated transfection, electroporation, genegun penetration, micro-injection, transposon/retrotransposon insertion,and adenoviral/retroviral/lentiviral infection.

Corresponding to the RGFP induction experiments shown above (FIGS. 4 and5), we also measure the expression of non-coding pre-miR-302s and itsmature miR-302 products in the pLenti-EF1alpha-RGFP-miR302-transformedbacteria with or without chemical induction. As shown in FIG. 6 andExample 4, the quantitative result of induced pre-miR-302s productionhas been confirmed by Northern blot analysis. Similar to the results ofthe RGFP induction experiments (FIGS. 4 and 5), the pre-miR-302sexpression is detected in transformed bacteria treated with MOPS,glycerin or ethanol, but not blank control, indicating that thesechemical pol-2 promoter inducers are also able to trigger non-coding RNAexpression in prokaryotic cells as well. Due to the structuralsimilarity of all microRNAs (miRNAs) and shRNAs, it is obvious for anordinary skill in the art to use other non-coding miRNAs and/or shRNAsin place of the miR-302 cluster for producing functional miRNAs, shRNAsand/or their precursors/homologs in prokaryotes.

Functional Application of the Present Invention in Stem Cell Generation

MicroRNA miR-302 has been used to reprogram mammalian somatic cells toembryonic stem cell (ESC)-like induced pluripotent stem (iPS) cells asdemonstrated in the U.S. patent application Ser. No. 12/149,725 and Ser.No. 12/318,806. Many stem cell applications and therapies have beendeveloped dependent on these ESC-like iPS cells. However, miR-302 isonly produced abundantly in human ESCs rather than other differentiatedtissue cells. Isolation of human ESCs is highly debatable. Cultivationof human ESCs is also very laborious and costly. Making syntheticmiR-302 mimics is an alternative way to bypass the need of human ESCs,yet still very expensive and inefficient. The similarity betweensynthetic and natural miR-302 is also questionable. To solve theseproblems, the present invention can provide a simple, cheap, fast andinducible composition and method for the bulk production of miR-302and/or its precursors/homologs in prokaryotes. Moreover, the isolationof miR-302 and/or its precursors from prokaryotic cells is relativelyeasy and cost-effective, as shown in FIG. 6 and Example 6 of the presentinvention.

We have used the pLenti-EF1alpha-RGFP-miR302-transformed E. coli cellsto produce and isolate high quantity and quality ofpLenti-EF1alpha-RGFP-miR302 vector and pre-miR-302s, as shown inExamples 5 and 6. Both pLenti-EF1alpha-RGFP-miR302 and pre-miR-302s areexpected to be useful for generating iPS cells in view of the U.S.patent application Ser. No. 12/149,725 and Ser. No. 12/318,806.Following Example 2, when the pre-miR-302s produced by the presentinvention are transduced into human skin primary keratinocytes, thetransfected keratinocytes are reprogrammed to ESC-like iPS cells andexpress strong ESC marker Oct4 (FIG. 7). Further shown in FIG. 8 andExample 8, bisulfite DNA sequencing assay also shows that global DNAdemethylation occurs in the promoter regions of Oct4 and Sox2 genes, twoof the most important reprogramming factors and ESC markers. As globalDNA demethylation and Oct4 expression are known to be the first stepthat cells have successfully begun the process of reprogramming toattain ESC-like pluripotency (Simonsson and Gurdon, Nat Cell Biol. 6:984-990, 2004), the miR-302 and/or pre-miR-302 isolated from theMOPS-induced bacterial extracts is proven to be effective for iPS cellgeneration. This example confirms that the present invention can be usedto prepare abundant miR-302 and/or pre-miR-302-containing bacterialextracts or lysates for pluripotent stem cell generation.

Application of microRNA Extracts in Tumor/Cancer Therapy

We may produce abundant microRNA miR-302 precursors (pre-miR-302s orpri-miR-302s) and the related miR-302-encoding plasmid vectors using thepresent invention (Examples 1, 5 and 6). The function and mechanism ofusing miR-302 in cancer therapy can be referred to the U.S. patentapplication Ser. No. 12/318,806 and Ser. No. 12/792,413. Our previousstudies have also demonstrated the feasibility of this approach intreating human melanoma, prostate cancer (Lin et al., RNA 2008), breastcancer, hepatocellular carcinoma, and embryonal teratocarcinoma cells(Lin et al., Cancer Res. 2010). As shown in FIGS. 9A-9B, all testedtumor/cancer cells were reprogrammed to normal iPS cells and formedembryoid body-like cell colonies after miR-302 and/or pre-miR-302treatment. The cells obtained after miR-302 and/or pre-miR-302 treatmentare labeled as mirPS cells, abbreviated for “miR302-induced pluripotentstem cells”. Moreover, miR-302 was also found to induce significantapoptosis (>95%) in all tested tumor/cancer cells but not normal tissuecells (Lin et al., RNA 2008 and Cancer Res. 2010). Flow cytometryanalysis comparing DNA content to cell cycle stages, further showed asignificant reduction in all mirPS mitotic cell populations (FIG. 9C).The mitotic cell population (M phase) was decreased by 78% from 49%±3%to 11%±2% in mirPS-MCF7, by 63% from 46%±4% to 17%±2% in mirPS-HepG2,and by 62% from 50%±6% to 19%±4% in mirPS-Tera2 cells, whereas theresting/dormant cell population (G0/G1 phase) was increased by 80% from41%±4% to 74%±5% in mirPS-MCF7, by 65% from 43%±3% to 71%±4% inmirPS-HepG2, and by 72% from 40%±7% to 69%±8% in mirPS-Tera2 cells,respectively. These results indicate that miR-302 and/or pre-miR-302 caneffectively attenuate the fast cell cycle rates and cause significantapoptosis in these tumor/cancer cells.

In vitro tumorigenicity assays, using Matrigel chambers (cell invasionassay, Example 9) and cell adhesion to the human bone marrow endothelialcell (hBMEC) monolayer (cell adhesion assay, Example 10), revealed twomore anti-tumorigenetic effects of miR-302 and/or pre-miR-302 inaddition to its anti-proliferative feature. Cell invasion assay showedthat all mirPS-tumor/cancer cells lost their ability to migrate (reducedto <1%) while the original tumor/cancer cells aggressively invaded intothe chambered areas supplemented with higher nutrients, representingover 9%±3% of MCF7, 16%±4% of Hep G2 and 3%±2% of Tera-2 cellpopulations (FIG. 10A). Consistently, cell adhesion assay also showedthat none of these mirPS-tumor/cancer cells could adhere to hBMECswhereas a significant population of original MCF7 (7%±3%) and Hep G2(20%±2%) cells quickly metastasize into the hBMEC monolayer after 50 minincubation (FIG. 10B). In sum, all of the findings thus far strongly andrepeatedly suggest that miR-302 and/or pre-miR-302 is a human tumorsuppressor capable of attenuating fast cell growth, causing tumor/cancercell apoptosis, and inhibiting tumor/cancer cell invasion as well asmetastasis. Most importantly, this novel miR-302 and/or pre-miR-302function may offer a universal treatment against multiple kinds of humancancers/tumors, including but not limited in malignant skin, prostate,breast, and liver cancers as well as various tumors in view of thevariety of different tissue types in embryonal teratomas.

After identifying the tumor suppressor function of miR-302 and/orpre-miR-302 and its different effects on normal and tumor/cancer cells,we have further tested the possible use of miR-302 and/or pre-miR-302 asan anti-cancer drug for treating Tera2-derived teratomas ineight-week-old male athymic mice (BALB/c nu/nu strain) (FIGS. 11A-11Cand Examples 11 and 12). Tera-2 cells are originally derived from humanembryonal teratocarcinomas that contain a variety of primitive tumoroustissue cells. Due to this pluripotent feature, Tera2-derived teratomasoften serve as a treatment model for various tumor types in vivo. Asshown in FIG. 11A, after miR-302 and/or pre-miR-302 treatment(Tera2+mir-302s) for three weeks, we detected a significant reduction ofthe average tumor size by >89% (11±5 mm³, n=6) compared to that ofnon-treated ones (104±23 mm³, n=4). In contrast, treating the sameamount of antisense-mir-302d (Terat2+mir-302d*) increased the averagetumor sizes by 140% (250±73 mm³, n=3). Northern blotting also showedthat miR-302 expression levels in these differently treated teratomacells negatively correlated to the tumor sizes (FIG. 11B), suggestingthat modulating miR-302 expression can effectively control the tumorgrowth in vivo. To validate these findings, we further performed westernblotting to confirm the co-suppression of G1-checkpoint regulators CDK2,cyclins-D1/D2 and BMI-1 and the co-activation of tumor suppressorsp16Ink4a and p14/p19Arf as well as core reprogramming factors Oct3/4,Sox2 and Nanog in the miR-302 and/or pre-miR-302-treated teratomas (FIG.11B). The same results were also confirmed by immunohistochemical (IHC)staining of these proteins in teratoma tissue sections (FIG. 11C). Basedon this novel tumor suppression function of miR-302 and the consistentdata in vitro and in vivo, it is conceivable that miR-302 can serve asan example microRNA for using the present invention in preparation ofdrugs for cancer therapy.

Application of microRNA Extracts for Wound Healing Treatment

We have tested the production of abundant microRNA miR-302 precursors(pre-miR-302s) and the related miR302-encoding plasmid vectors for usein a wound healing animal trial, using the present invention (Example13). The pre-miR-302s and their related plasmid vectors were amplifiedas described in Examples 1 and 5 and extracted as described in Examples5 and 6. Then, the isolated pre-miR-302s and their related plasmidvectors were mixed with a pre-prepared ointment base containing cocoabutter, cottonseed oil, olive oil, sodium pyruvate, and whitepetrolatum. The concentration of miR-302 precursors and vectors in theprepared ointment base is 10 μg/mL. Skin open wounds were generated byscalpel dissection. Ointment with or without miR-302 and/or pre-miR-302was directly applied on the wound, respectively, and covered the wholewounded area. Then, the treated area was further sealed by liquidbandage. As show in FIG. 12, within two weeks, the results clearlyshowed that miR-302 and/or pre-miR-302 treatments significantly enhancedthe speed of wound healing over twice faster than all other treatmentsand controls. Moreover, miR-302 and/or pre-miR-302-treated wound healingarea showed normal hair regrowth and left no scar, while othertreatments resulted in minor scar tissues with no hair (as indicated byblack arrow).

Other Applications of the Present Invention

One preferred application embodiment of the present invention is togenerate microRNAs and/or shRNAs for biomedical research, pharmaceuticaland therapeutic applications, such as gene modulation and gene therapy.For example, but not limited, miR-302 has been found to be a tumorsuppressor in human cells, as demonstrated in the U.S. patentapplication Ser. No. 12/318,806. The present invention may be used toproduce abundant miR-302 and/or its precursors/homologs for cancertherapy or drug development. Particularly, a therapeutic microRNA/shRNAgene isolated from a microbe, plant or animal can be cloned into aplasmid vector under the control of a eukaryotic pol-2 or pol-2compatible viral promoter and delivered into non-pathogenic bacteria.When the bacteria containing such a microRNA/shRNA expression vector isintroduced into patient's cells, the patient can drink glycerin orethanol to trigger the generation and release of the therapeuticmicroRNA/shRNA from the bacteria into the cells, so as to cure thedisorders and/or diseases.

Another preferred application embodiment of the present invention is togenerate functional proteins/peptides for biomedical research,pharmaceutical and therapeutic applications. For example, but notlimited, the protein/peptide so obtained can be insulin for treatingDiabetes, tumor suppressor proteins for curing tumors/cancers, growthfactors for stimulating normal body development, antibodies forbiomedical research or vaccine/serum production, and all varieties ofbiological enzymes for molecular biology and biomedical research.Particularly, a therapeutic protein/peptide gene isolated from amicrobe, plant or animal can be cloned into a plasmid vector under thecontrol of a eukaryotic pol-2 or pol-2 compatible viral promoter anddelivered into non-pathogenic bacteria. When the bacteria containingsuch a gene vector is introduced into patient's cells, the patient candrink glycerin or ethanol to trigger the expression and release of thetherapeutic protein/peptide from the bacteria into the cells, so as tocure the disorders and/or diseases.

Another one preferred application embodiment of the present invention isto generate high-yield protein food and drug supplies for humans and/oranimals. Protein production by fast growing bacteria can reduce the timeand labor for maintaining expensive animal stocks. Also, we can preventunnecessary animal sacrifices. Advantageously, the present invention caneven produce mammalian proteins using mammalian gene promoters, reducingthe risks of genetic engineering and gene modification.

Another one possible but not preferred application embodiment of thepresent invention is to make biological weapons. For example, apoison/toxic protein gene isolated from a microbe, plant or animal,including but not limited to, Bacillus anthracis, poison ivy, jellyfish,insect, fish, amphibian, and snake, can be cloned into a plasmid vectorunder the control of a eukaryotic pol-2 or pol-2 compatible viralpromoter and delivered into non-pathogenic bacteria. When there is nochemical agent that can induce pol-2 promoter-driven gene expression,these bacteria carrying such a plasmid vector will be stealthilyamplified and present no harm to anyone. However, once there is achemical agent like MOPS, ethanol or glycerin, or a mixture thereof,presented in the environment, the poison/toxic gene in the bacteria isthen activated to manifest its effects.

DEFINITION, COMPOSITION AND APPLICATIONS A. Definitions

To facilitate understanding of the invention, a number of terms aredefined below:

Nucleic Acid: a polymer of deoxyribonucleic acid (DNA) or ribonucleicacid (RNA), either single or double stranded.

Nucleotide: a monomeric unit of DNA or RNA consisting of a sugar moiety(pentose), a phosphate, and a nitrogenous heterocyclic base. The base islinked to the sugar moiety via the glycosidic carbon (1′ carbon of thepentose) and that combination of base and sugar is a nucleoside. Anucleoside containing at least one phosphate group bonded to the 3′ or5′ position of the pentose is a nucleotide. DNA and RNA are consisted ofdifferent types of nucleotide units called deoxyribonucleotide andribonucleotide, respectively.

Oligonucleotide: a molecule comprised of two or more monomeric units ofDNA and/or RNA, preferably more than three, and usually more than ten.An oligonucleotide longer than 13 nucleotide monomers is also calledpolynucleotide. The exact size will depend on many factors, which inturn depends on the ultimate function or use of the oligonucleotide. Theoligonucleotide may be generated in any manner, including chemicalsynthesis, DNA replication, RNA transcription, reverse transcription, ora combination thereof.

Nucleotide Analog: a purine or pyrimidine nucleotide that differsstructurally from adenine (A), thymine (T), guanine (G), cytosine (C),or uracil (U), but is sufficiently similar to substitute for the normalnucleotide in a nucleic acid molecule.

Nucleic Acid Composition: a nucleic acid composition refers to anoligonucleotide or polynucleotide such as a DNA or RNA sequence, or amixed DNA/RNA sequence, in either a single-stranded or a double-strandedmolecular structure.

Gene: a nucleic acid composition whose oligonucleotide or polynucleotidesequence codes for an RNA and/or a polypeptide (protein). A gene can beeither RNA or DNA. A gene may encode a non-coding RNA, such as smallhairpin RNA (shRNA), microRNA (miRNA), rRNA, tRNA, snoRNA, snRNA, andtheir RNA precursors as well as derivatives. Alternatively, a gene mayencode a protein-coding RNA essential for protein/peptide synthesis,such as messenger RNA (mRNA) and its RNA precursors as well asderivatives. In some cases, a gene may encode a protein-coding RNA thatalso contains at least a microRNA or shRNA sequence.

Primary RNA Transcript: an RNA sequence that is directly transcribedfrom a gene without any RNA processing or modification.

Precursor messenger RNA (pre-mRNA): primary RNA transcripts of aprotein-coding gene, which are produced by eukaryotic type-II RNApolymerase (Pol-II) machineries in eukaryotes through an intracellularmechanism termed transcription. A pre-mRNA sequence contains a5′-untranslated region (UTR), a 3′-UTR, exons and introns.

Intron: a part or parts of a gene transcript sequence encodingnon-protein-reading frames, such as in-frame intron, 5′-UTR and 3′-UTR.

Exon: a part or parts of a gene transcript sequence encodingprotein-reading frames (cDNA), such as cDNA for cellular genes, growthfactors, insulin, antibodies and their analogs/homologs as well asderivatives.

Messenger RNA (mRNA): assembly of pre-mRNA exons, which is formed afterintron removal by intracellular RNA splicing machineries (e.g.spliceosomes) and served as a protein-coding RNA for peptide/proteinsynthesis. The peptides/proteins encoded by mRNAs include, but notlimited, enzymes, growth factors, insulin, antibodies and theiranalogs/homologs as well as derivatives.

Complementary DNA (cDNA): a single-stranded or double-stranded DNA thatcontains a sequence complementary to an mRNA sequence and does notcontain any intronic sequence.

Sense: a nucleic acid molecule in the same sequence order andcomposition as the homologous mRNA. The sense conformation is indicatedwith a “+”, “s” or “sense” symbol.

Antisense: a nucleic acid molecule complementary to the respective mRNAmolecule. The antisense conformation is indicated as a “−” symbol orwith an “a” or “antisense” in front of the DNA or RNA, e.g., “aDNA” or“aRNA”.

Base Pair (bp): a partnership of adenine (A) with thymine (T), or ofcytosine (C) with guanine (G) in a double stranded DNA molecule. In RNA,uracil (U) is substituted for thymine. Generally the partnership isachieved through hydrogen bonding. For example, a sense nucleotidesequence “5′-A-T-C-G-U-3” can form complete base pairing with itsantisense sequence “5′-A-C-G-A-T-3”.

5′-end: a terminus lacking a nucleotide at the 5′ position of successivenucleotides in which the 5′-hydroxyl group of one nucleotide is joinedto the 3′-hydroxyl group of the next nucleotide by a phosphodiesterlinkage. Other groups, such as one or more phosphates, may be present onthe terminus.

3′-end: a terminus lacking a nucleotide at the 3′ position of successivenucleotides in which the 5′-hydroxyl group of one nucleotide is joinedto the 3′-hydroxyl group of the next nucleotide by a phosphodiesterlinkage. Other groups, most often a hydroxyl group, may be present onthe terminus.

Template: a nucleic acid molecule being copied by a nucleic acidpolymerase. A template can be single-stranded, double-stranded orpartially double-stranded, depending on the polymerase. The synthesizedcopy is complementary to the template, or to at least one strand of adouble-stranded or partially double-stranded template. Both RNA and DNAare synthesized in the 5′ to 3′ direction. The two strands of a nucleicacid duplex are always aligned so that the 5′ ends of the two strandsare at opposite ends of the duplex (and, by necessity, so then are the3′ ends).

Nucleic Acid Template: a double-stranded DNA molecule, double strandedRNA molecule, hybrid molecules such as DNA-RNA or RNA-DNA hybrid, orsingle-stranded DNA or RNA molecule.

Conserved: a nucleotide sequence is conserved with respect to apre-selected (referenced) sequence if it non-randomly hybridizes to anexact complement of the pre-selected sequence.

Homologous or Homology: a term indicating the similarity between apolynucleotide and a gene or mRNA sequence. A nucleic acid sequence maybe partially or completely homologous to a particular gene or mRNAsequence, for example. Homology may be expressed as a percentagedetermined by the number of similar nucleotides over the total number ofnucleotides.

Complementary or Complementarity or Complementation: a term used inreference to matched base pairing between two polynucleotides (i.e.sequences of an mRNA and a cDNA) related by the aforementioned “basepair (bp)” rules. For example, the sequence “5′-A-G-T-3” iscomplementary to the sequence “5′-A-C-T-3”, and also to “5′-A-C-U-3”.Complementation can be between two DNA strands, a DNA and an RNA strand,or between two RNA strands. Complementarity may be “partial” or“complete” or “total”. Partial complementarity or complementation occurswhen only some of the nucleic acid bases are matched according to thebase pairing rules. Complete or total complementarity or complementationoccurs when the bases are completely or perfectly matched between thenucleic acid strands. The degree of complementarity between nucleic acidstrands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in amplification reactions, as well as in detection methodsthat depend on binding between nucleic acids. Percent complementarity orcomplementation refers to the number of mismatch bases over the totalbases in one strand of the nucleic acid. Thus, a 50% complementationmeans that half of the bases were mismatched and half were matched. Twostrands of nucleic acid can be complementary even though the two strandsdiffer in the number of bases. In this situation, the complementationoccurs between the portion of the longer strand corresponding to thebases on that strand that pair with the bases on the shorter strand.

Complementary Bases: nucleotides that normally pair up when DNA or RNAadopts a double stranded configuration.

Complementary Nucleotide Sequence: a sequence of nucleotides in asingle-stranded molecule of DNA or RNA that is sufficientlycomplementary to that on another single strand to specifically hybridizebetween the two strands with consequent hydrogen bonding.

Hybridize and Hybridization: the formation of duplexes betweennucleotide sequences which are sufficiently complementary to formcomplexes via base pairing. Where a primer (or splice template)“hybridizes” with target (template), such complexes (or hybrids) aresufficiently stable to serve the priming function required by a DNApolymerase to initiate DNA synthesis. There is a specific, i.e.non-random, interaction between two complementary polynucleotides thatcan be competitively inhibited.

Posttranscriptional Gene Silencing: a targeted gene knockout orknockdown effect at the level of mRNA degradation or translationalsuppression, which is usually triggered by either foreign/viral DNA orRNA transgenes or small inhibitory RNAs.

RNA Interference (RNAi): a posttranscriptional gene silencing mechanismin eukaryotes, which can be triggered by small inhibitory RNA moleculessuch as microRNA (miRNA), small hairpin RNA (shRNA) and smallinterfering RNA (siRNA). These small RNA molecules usually function asgene silencers, interfering with expression of intracellular genescontaining either completely or partially complementarity to the smallRNAs.

Gene Silencing Effect: a cell response after a gene function issuppressed, consisting but not limited of cell cycle attenuation,G0/G1-checkpoint arrest, tumor suppression, anti-tumorigenicity, cancercell apoptosis, and a combination thereof.

Non-coding RNA: an RNA transcript that cannot be used to synthesizepeptides or proteins through intracellular translation machineries.Non-coding RNA includes long and short regulatory RNA molecules such asmicroRNA (miRNA), small hairpin RNA (shRNA), small interfering RNA(siRNA) and double strand RNA (dsRNA). These regulatory RNA moleculesusually function as gene silencers, interfering with expression ofintracellular genes containing either completely or partiallycomplementarity to the non-coding RNAs.

MicroRNA (miRNA): single-stranded RNAs capable of binding to targetedgene transcripts that have partial complementarity to the miRNA. MiRNAis usually about 17-27 oligonucleotides in length and is able to eitherdirectly degrade its intracellular mRNA target(s) or suppress theprotein translation of its targeted mRNA, depending on thecomplementarity between the miRNA and its target mRNA. Natural miRNAsare found in almost all eukaryotes, functioning as a defense againstviral infections and allowing regulation of gene expression duringdevelopment of plants and animals.

Precursor MicroRNA (Pre-miRNA): hairpin-like single-stranded RNAscontaining stem-arm and stem-loop regions for interacting withintracellular RNaseIII endoribonucleases to produce one or multiplemicroRNAs (miRNAs) capable of silencing a targeted gene or genescomplementary to the microRNA sequence(s). The stem-arm of a pre-miRNAcan form either a perfectly (100%) or a partially (mis-matched) hybridduplexes, while the stem-loop connects one end of the stem-arm duplex toform a circle or hairpin-loop conformation. In the present invention,however, precursor of microRNA may also includes pri-miRNA.

Small interfering RNA (siRNA): short double-stranded RNAs sized about18-27 perfectly base-paired ribonucleotide duplexes and capable ofdegrading target gene transcripts with almost perfect complementarity.

Small or short hairpin RNA (shRNA): single-stranded RNAs that contain apair of partially or completely matched stem-arm nucleotide sequencesdivided by an unmatched loop oligonucleotide to form a hairpin-likestructure. Many natural miRNAs are derived from hairpin-like RNAprecursors, namely precursor microRNA (pre-miRNA).

Vector: a recombinant nucleic acid composition such as recombinant DNA(rDNA) capable of movement and residence in different geneticenvironments. Generally, another nucleic acid is operatively linkedtherein. The vector can be capable of autonomous replication in a cellin which case the vector and the attached segment is replicated. Onetype of preferred vector is an episome, i.e., a nucleic acid moleculecapable of extrachromosomal replication. Preferred vectors are thosecapable of autonomous replication and expression of nucleic acids.Vectors capable of directing the expression of genes encoding for one ormore polypeptides and/or non-coding RNAs are referred to herein as“expression vectors” or “expression-competent vectors”. Particularlyimportant vectors allow cloning of cDNA from mRNAs produced using areverse transcriptase. A vector may contain components consisting of aviral or a type-II RNA polymerase (Pol-II or pol-2) promoter, or both, aKozak consensus translation initiation site, polyadenylation signals, aplurality of restriction/cloning sites, a pUC origin of replication, aSV40 early promoter for expressing at least an antibiotic resistancegene in replication-competent prokaryotic cells, an optional SV40 originfor replication in mammalian cells, and/or a tetracycline responsiveelement. The structure of a vector can be a linear or circular form ofsingle- or double-stranded DNA selected form the group consisting ofplasmid, viral vector, transposon, retrotransposon, DNA transgene,jumping gene, and a combination thereof.

Promoter: a nucleic acid to which a polymerase molecule recognizes,perhaps binds to, and initiates RNA transcription. For the purposes ofthe instant invention, a promoter can be a known polymerase bindingsite, an enhancer and the like, any sequence that can initiate synthesisof RNA transcripts by a desired polymerase.

Eukaryotic Promoter: a sequence of nucleic acid motifs which arerequired for gene transcription and can be recognized by eukaryotic typeII RNA polymerases (pol-2), pol-2 equivalent, and/or pol-2 compatibleviral polymerases

Type-II RNA Polymerase (Pol-II or pol-2) Promoter: a RNA promoter thatis recognized and bound by eukaryotic type-II RNA polymerases (Pol-II orpol-2) which transcribe eukaryotic messenger RNAs (mRNAs) and/ormicroRNAs (miRNAs). For example, but not limited, a pol-2 promoter canbe a mammalian RNA promoter or a cytomegaloviral (CMV) promoter.

Type-II RNA Polymerase (Pol-II or pol-2) Equivalent: an eukaryotictranscription machinery selected from the group consisting of mammaliantype-II RNA polymerases (Pol-II or pol-2) and Pol-II compatible viralRNA polymerases.

Pol-II Compatible Viral Promoter: a viral RNA promoter capable of usingthe eukaryotic poi-2 or equivalent transcription machinery for its geneexpression. For example, but not limited, a pol-2 compatible viralpromoter can be a cytomegaloviral (CMV) promoter or a retroviral longterminal repeat (LTR) promoter.

Cistron: a sequence of nucleotides in a DNA molecule coding for an aminoacid residue sequence and including upstream and downstream DNAexpression control elements.

RNA Processing: a cellular mechanism responsible for RNA maturation,modification and degradation, including RNA splicing, intron excision,exosome digestion, nonsense-mediated decay (NMD), RNA editing, RNAprocessing, and a combination thereof.

Antibiotic Resistance Gene: a gene capable of degrading antibioticsselected from the group consisted of penicillin G, streptomycin,ampicillin (Amp), neomycin, G418, kanamycin, erythromycin, paromycin,phophomycin, spectromycin, tetracycline (Tet), doxycycline (Dox),rifapicin, amphotericin B, gentamycin, chloramphenicol, cephalothin,tylosin, and a combination thereof.

Restriction/Cloning Site: a DNA motif for restriction enzyme cleavageincluding but not limited to AatII, AccI, AflII/III, AgeI, ApaI/LI,AseI, Asp718I, BamHI, BbeI, BclI/II, BglII, BsmI, Bsp120I,BspHI/LU11I/120I, BsrI/BI/GI, BssHII/SI, BstBI/U1/XI, ClaI, Csp6I, DpnI,DraI/II, EagI, Ecl136II, EcoRI/RII/47III/RV, EheI, FspI, HaeIII, HhaI,HinPI, HindIII, HinfI, HpaI/II, KasI, KpnI, MaeII/III, MfeI, MluI, MscI,MseI, NaeI, NarI, NcoI, NdeI, NgoMI, NotI, NruI, NsiI, PmlI, Ppu10I,PstI, PvuI/II, RsaI, SacI/II, SalI, Sau3AI, SmaI, SnaBI, SphI, SspI,StuI, TaiI, TaqI, XbaI, XhoI, XmaI cleavage site.

Gene Delivery: a genetic engineering method selected from the groupconsisting of polysomal transfection, liposomal transfection, chemicaltransfection, electroporation, viral infection, DNA recombination,transposon insertion, jumping gene insertion, microinjection, gene-gunpenetration, and a combination thereof.

Genetic Engineering: a DNA recombination method selected from the groupconsisting of DNA restriction and ligation, homologous recombination,transgene incorporation, transposon insertion, jumping gene integration,retroviral infection, and a combination thereof.

Cell Cycle Regulator: a cellular gene involved in controlling celldivision and proliferation rates, consisting but not limited of CDK2,CDK4, CDK6, cyclins, BMI-1, p14/p19Arf, p15Ink4b, p16Ink4a, p18Ink4c,p21Cip1/Waf1, and p27Kip1, and a combination thereof.

Tumor Suppression: a cellular anti-tumor and anti-cancer mechanismconsisting but not limited of cell cycle attenuation, G0/G1-checkpointarrest, tumor suppression, anti-tumorigenicity, cancer cell apoptosis,and a combination thereof.

Targeted Cell: a single or a plurality of human cells selected from thegroup consisting of a somatic cell, a tissue, a stem cell, a germ-linecell, a teratoma cell, a tumor cell, a cancer cell, and a combinationthereof.

Cancerous Tissue: a neoplastic tissue derived from the group consistingof skin cancer, prostate cancer, breast cancer, liver cancer, lungcancer, brain tumor/cancer, lymphoma, leukemia and a combinationthereof.

Transcription Inducer: a chemical agent that can stimulate and/orenhance RNA transcription from a gene. A transcription inducer contains,but not limited, a chemical structure similar to3-morpholinopropane-1-sulfonic acid (MOPS), ethanol or glycerin, or amixture thereof.

Antibody: a peptide or protein molecule having a pre-selected conserveddomain structure coding for a receptor capable of binding a pre-selectedligand.

Pharmaceutical or therapeutic Application: a biomedical utilizationand/or apparatus useful for stem cell generation, stem cell researchand/or therapy development, cancer therapy, disease treatment, woundhealing treatment, high-yield production of drug and/or food supplies,and a combination thereof.

B. Compositions and Applications

A composition and its use for inducing RNA and/or protein expression inprokaryotes, comprising: (a) at least a chemical agent containing astructure similar to 3-morpholinopropane-1-sulfonic acid (MOPS), ethanolor glycerin, or a mixture thereof; and (b) a plurality of prokaryoticcells that contain at least a gene mediated by a eukaryotic pol-2promoter-driven or a pol-2 compatible viral promoter-driven expressionmechanism; wherein (a) and (b) are mixed together under a condition toinduce the expression of said gene, so as to generate the RNA and/orprotein products of said gene.

Alternatively, the present invention is an inducible gene expressioncomposition using chemical agents to stimulate eukaryotic RNApromoter-driven transcription in prokaryotes. An inducible geneexpression composition, comprising (a) at least a chemical agentcontaining a structure similar to 3-morpholinopropane-1-sulfonic acid(MOPS), ethanol or glycerin, or a mixture thereof; and (b) a pluralityof prokaryotic cells that contain at least a gene mediated by aeukaryotic pol-2 promoter-driven or a pol-2 compatible viralpromoter-driven expression mechanism; wherein (a) and (b) are mixedtogether under a condition to induce the expression of said gene.

In principle, the present invention provides a novel composition designand its applicable strategy for inducing a quick adaptation ofprokaryotes to use eukaryotic pol-2 promoters for directly expressingRNAs and/or proteins of interest without the need of changing toerror-prone prokaryotic promoters or growing laborious and costlyhybridomas or mammalian cells.

Preferably, said prokaryote is a bacterial cells in particular,Escherichia coli (E. coli), and said chemical agent is3-morpholinopropane-1-sulfonic acid (MOPS), ethanol or glycerin, or amixture thereof. Also preferably, said eukaryotic RNA promoter is eithera eukaryotic pol-2 promoter, such as EF1alpha, or a pol-2 compatibleviral promoter, such as cytomegaloviral (CMV) promoter or retrovirallong terminal repeat (LTR) promoter. The gene mediated by saideukaryotic RNA promoter is coded for either a non-coding or aprotein-coding RNA transcript, or both, selected from the groupconsisted of microRNA (miRNA), small hairpin RNA (shRNA), smallinterfering RNA (siRNA), messenger RNA (mRNA), their precursors andhomologs, and a combination thereof. The peptide/protein generated bythe present invention is translated from the above protein-coding mRNAtranscripts and may be selected from, but not limited to, the groupconsisted of enzyme, growth factor, antibody, insulin, botulinum toxin(botox), a functional protein and its homologs/analogs, and acombination thereof. Preferably, said condition for inducing theexpression of said gene is a bacterial culturing condition inLuria-Bertani (LB) broth at 37° C. with the addition of said chemicalagents.

Recap of the Drawing Illustrations:

Referring particularly to the drawings for the purpose of illustrationonly and not limitation, there is illustrated:

Referring to FIGS. 1A and 1B, there is shown an inducible pol-2promoter-driven gene expression composition (A) and its mechanisms (B)for RNA transcript and protein production in prokaryotes and eukaryotesand microRNA (miRNA) production in eukaryotes. For demonstrating thepresent invention, pLenti-EF1alpha-RGFP-miR302 is served as an examplecomposition to transform competent E. coli DH5alpha cells for producingRGFP mRNA and protein as well as miR-302 miRNA and/or precursors thereofunder the control of MOPS, glycerin and/or ethanol induction. ThepLenti-EF1alpha-RGFP-miR302 is a lentiviral plasmid vector that isdesigned to expresses various miRNAs/shRNAs, mRNAs and/orproteins/peptides in both prokaryotes and eukaryotes. According to thedisclosed mechanism (B), it is possible for an ordinary skill in the artto use any microRNA/shRNA in place miR-302 or any mRNA/protein in placeof RGFP as taught in the present invention. Black arrows indicate thepathways occurring in both prokaryotic and eukaryotic cells, while blankarrows indicate the steps only occurring in the eukaryotic cells.

Referring to FIG. 2, there is depicted the results of bacterial culturebroths treated with (left) or without (right) the mixture of 0.1% (v/v)MOPS and 0.05% (v/v) glycerin. The E. coli bacteria have beentransformed by pLenti-EF1alpha-RGFP-miR302 before treatments.

Referring to FIG. 3, there is shown the results of different bacterialpellets after treated with 0.1% (v/v) MOPS. The E. coli bacteria havebeen transformed by either pLVX-Gm-miR302+367 (green) orpLenti-EF1alpha-RGFP-miR302 (red) before MOPS treatment.

Referring to FIG. 4, there is shown the inducibility of differentchemicals for inducing pol-2 promoter-driven gene expression incompetent E. coli DH5alpha cells. Among all chemicals tested in thepresent invention, the top three most potent inducers are MOPS, glycerinand ethanol. The chemical concentration used can be ranged from 0.001%to 4%, most preferably, from 0.01 to 1%.

Referring to FIG. 5, there is shown the Western blotting results of redRGFP protein expression induced by MOPS, glycerin and ethanol,respectively. Bacterial RuvB protein is used as a house-keeping standardto normalize the RGFP expression. Protein extraction from blank E. coliDH5alpha cells, i.e. transformed no vectors, serves as a negativecontrol.

Referring to FIG. 6, there is shown the Northern blotting results ofmiR-302 and its pre-miRNA cluster expression induced by MOPS, glycerinand ethanol, respectively. RNA extraction from blank E. coli DH5alphacells serves as a negative control.

Referring to FIG. 7, there is shown iPS cell generation using miR-302and/or pre-miR-302 isolated from bacterial extracts (BE), which isconfirmed by Northern blot analysis as shown in FIG. 6. As previouslyreported, miR-302-reprogrammed iPS cells (mirPSCs) form sphere-like cellcolonies and express strong Oct4, a standard ESC marker.

Referring to FIG. 8, there is shown the global DNA demethylation of Oct4and Sox2 gene promoters induced by the miR-302 and/or pre-miR-302isolated from bacterial extracts (BE), which is confirmed by Northernblot analysis as shown in FIG. 6. As demonstrated by Simonsson andGurdon (Nat Cell Biol. 6, 984-990, 2004), both signs of global DNAdemethylation and Oct4 expression are required for somatic cellreprogramming.

Referring to FIGS. 9A-9C and 10A-10B, there is shown in vitrotumorigenicity assays of various tumor/cancer cells in response tomiR-302 and/or pre-miR-302 treatment. The cells obtained after miR-302and/or pre-miR-302 treatment are labeled as mirPS cells, includingbreast cancer-derived mirPS-MCF7, liver cancer-derived mirPS-HepG2, andembryonal teratocarcinoma-derived mirPS-Tera2 cells. FIGS. 9A-9B:Changes of cell morphology and cell cycle rate before and after miR-302and/or pre-miR-302 treatment. Each cell DNA content respective to cellcycle stages was shown by a chart of flow cytometry analysis above thecell morphology (n=3, p<0.01). FIG. 9C: Bar charts of flow cytometryanalyses showing the dose-dependent miR-302 and/or pre-miR-302 effect onthe changes of mitotic (M phase) and dormant (G0/G1 phase) cellpopulations of various treated tumor/cancer cells. FIG. 10A: Functionalanalysis of miR-302-suppressed tumor invasion in Matrigel chambers (n=4,p<0.05). FIG. 10B: Comparison of cell adhesion to the hBMEC monolayerbefore and after miR-302 and/or pre-miR-302 treatment (n=4, p<0.05).

Referring to FIGS. 11A-11C, there is shown in vivo tumorigenicity assaysof embryonal teratocarcinoma Tera-2 cells in response to either thewhole miR-302 familial cluster (Tera2+mir-302s) or antisense miR-302donly (Tera2+mir-302d*) treatment (n=3, p<0.05). Embryonalteratocarcinoma often contains various tumor/cancer cells derived fromall three embryonic germ layers (i.e. ectoderm, mesoderm and endoderm),which represent a mixed neoplastic tissue useful for testinganti-tumor/cancer drugs. (A) Morphological evaluation of average tumorsizes three weeks after the in-situ injection (post-is). All tumors werelocalized in the original implant sites (black arrows). No signs ofcachexia or tumor metastasis were observed in all tested mice. (B)Northern and western blot analyses and (C) Immunohistochemical staininganalyses of the in vivo miR-302 effect on the expression patterns ofcore reprogramming factors Oct3/4-Sox2-Nanog and miR-302-targetedG1-checkpoint regulators CDK2, cyclins D1/D2 and BMI-1 as well asp16Ink4a and p14Arf.

Referring to FIG. 12, there is shown the results of an in-vivo woundhealing trial using microRNA miR-302 and/or pre-miR-302 containingointment to treat skin open wounds in mice. The skin wounds of miR-302and/or pre-miR-302-treated mice were at least twice larger than those ofcontrol mice treated with only blank ointment or other microRNA(miR-HA). The trial results clearly demonstrated that miR-302 and/orpre-miR-302 treatments significantly enhanced the speed of wound healingover two times faster than all other treatments and controls. Moreover,miR-302 and/or pre-miR-302-treated wound healing area showed normal hairregrowth and left no scar while other treatments resulted in minor scarswith no hair (indicated by black arrow).

EXAMPLES 1. Bacterial Cell Culture and Chemical Treatments

Competent E. coli DH5alpha cells are acquired from the z-competent E.coli transformation kit (Zymo Research, Irvine, Calif.) and transformedby mixing with 5 μg of a desired plasmid vector such aspLVX-Gm-miR302+367 or pLenti-EF1alpha-RGFP-miR302. Non-transformedbacterial cells are normally grown in Luria-Bertani (LB) brothsupplemented with 10 mM MgSO₄ and 0.2 mM glucose at 37° C. with frequentagitation at 170 rpm, whereas the transformed bacterial cells arecultivated in the above LB broth further supplemented with additional100 ampicillin. For chemical induction, 0.5 to 2 ml of MOPS, glycerin,and ethanol, respectively or in combination, is added into 1 litter LBbroth supplemented with 10 mM MgSO₄ and 0.2 mM glucose in the presenceof 100 μg/ml ampicillin. For negative control, the transformed bacterialcells are cultivated in the above ampicillin-supplemented LB broth butwithout out adding any chemical inducer.

2. Human Cell Culture and MicroRNA Transfection

Human primary epidermal skin cells (hpESCs) are isolated and dissociatedfrom a minimum of 2 cubic mm by 4 mg/ml collagenase I digestion at 37°C. for 35 min in fresh RPMI 1640 medium supplemented with 20% FBS. Forculturing keratinocytes, the isolated cells are cultivated in EpiLifeserum-free cell culture medium supplemented with human keratinocytegrowth supplements (HKGS, Invitrogen, Carlsbad, Calif.) in the absenceof antibiotics at 37° C. under 5% CO₂. Culture cells are passaged at50%-60% confluency by exposing cells to trypsin/EDTA solution for 1 minand rinsing once with phenol red-free DMEM medium (Invitrogen), and thedetached cells are replated at 1:10 dilution in fresh EpiLife mediumwith HKGS supplements. Human cancer/tumor cell lines MCF7, HepG2 andTera-2 were obtained from the American Type Culture Collection (ATCC,Rockville, Md.) and maintained according to manufacturer's suggestions.For microRNA transfection, 15 μg of isolated miR-302 and/or precursorthereof is dissolved in 1 ml of fresh EpiLife medium and mixed with 50μl of X-tremeGENE HP DNA transfection reagent. After 10 min incubation,the mixture is added into a 100-mm cell culture dish containing 50%-60%confluency of hpESCs or the cancer/tumor cells, respectively. The mediumis replaced by fresh EpiLife medium with HKGS supplements or theconditioned medium suggested by ATCC 12 to 18 hours later. Thistransfection procedure may be repeated 3 to 4 times every three-fourdays to increase transfection efficiency. After cell morphology becomesphere-like, the cells (mirPSCs) are grown and passaged in knockoutDMEM/F-12 medium (Invitrogen) supplemented with 20% knockout serum, 1%MEM nonessential amino acids, 100 μM β-mercaptoethanol, 1 mM GlutaMax, 1mM sodium pyruvate, 10 ng/ml bFGF, 10 ng/ml FGF-4, 5 ng/ml LIF, 100IU/ml penicillin/100 μg/ml streptomycin, 0.1 μM A83-01, and 0.1 μMvalproic acid (Stemgent, San Diego, Calif.), at 37° C. under 5% CO₂.

3. Protein Extraction and Western Blot Analysis

Cells (10⁶) are lysed with a CelLytic-M lysis/extraction reagent (Sigma)supplemented with protease inhibitors, Leupeptin, TLCK, TAME and PMSF,following the manufacturer's suggestion. Lysates are centrifuged at12,000 rpm for 20 min at 4° C. and the supernatant is recovered. Proteinconcentrations are measured using an improved SOFTmax protein assaypackage on an E-max microplate reader (Molecular Devices, CA). Each 30μg of cell lysate are added to SDS-PAGE sample buffer under reducing(+50 mM DTT) and non-reducing (no DTT) conditions, and boiled for 3 minbefore loading onto a 6˜8% polyacrylamide gel. Proteins are resolved bySDS-polyacrylamide gel electrophoresis (PAGE), electroblotted onto anitrocellulose membrane and incubated in Odyssey blocking reagent(Li-Cor Biosciences, Lincoln, NB) for 2 hours at room temperature. Then,a primary antibody is applied to the reagent and incubated the mixtureat 4° C. Primary antibodies include Oct3/4 (Santa Cruz Biotechnology,Santa Cruz, Calif.), Sox2 (Santa Cruz), Nanog (Santa Cruz), CDK2 (SantaCruz), cyclin D1 (Santa Cruz), cyclin D2 (Abcam), BMI-1 (Santa Cruz),keratin 16 (Abcam), β-actin (Chemicon, Temecula, Calif.), RuvB (SantaCruz) and RGFP (Clontech). After overnight, the membrane is rinsed threetimes with TBS-T and then exposed to goat anti-mouse IgG conjugatedsecondary antibody to Alexa Fluor 680 reactive dye (1:2,000;Invitrogen-Molecular Probes), for 1 hour at the room temperature. Afterthree additional TBS-T rinses, fluorescent scanning of the immunoblotand image analysis are conducted using Li-Cor Odyssey Infrared Imagerand Odyssey Software v.10 (Li-Cor).

4. RNA Extraction and Northern Blot Analysis

Total RNAs (10 μg) are isolated with a mirVana™ miRNA isolation kit(Ambion, Austin, Tex.), fractionated by either 15% TBE-ureapolyacrylamide gel or 3.5% low melting point agarose gelelectrophoresis, and electroblotted onto a nylon membrane. Detection ofmiR-302 and/or pre-miR-302 is performed with a [LNA]-DNA probe(5′-[TCACTGAAAC] ATGGAAGCAC TTA-3′) (SEQ.ID.NO.1) probe. The probe hasbeen purified by high-performance liquid chromatography (HPLC) andtail-labeled with terminal transferase (20 units) for 20 min in thepresence of [³²P]-dATP (>3000 Ci/mM, Amersham International, ArlingtonHeights, Ill.).

5. Plasmid Amplification and Plasmid DNA/Total RNA Extraction

Competent E. coli DH5alpha cells treated with plasmid transformation(from Example 1) are cultivated overnight in LB broth supplemented with10 mM MgSO₄ and 0.2 mM glucose at 37° C. with frequent agitation at 170rpm. For inducing eukaryotic promoter-driven RNA and/or proteinproduction, 0.5 to 2 ml of MOPS, glycerin, and/or ethanol is added intoevery 1 litter of LB broth for the above bacterial cultivation andamplification. All amplified plasmid DNAs and expressed mRNAs/microRNAsare isolated together using a HiSpeed plasmid purification kit (Qiagen,Valencia, Calif.), following the manufacturer's protocol but with aminor modification that RNase A is not added into the P1 buffer. Thefinal extracted products containing both plasmids and mRNAs/microRNAsare dissolved in DEPC-treated ddH₂O and stored at −80° C. before use.For purifying only the amplified plasmid vectors, RNase A is added intothe P1 buffer and the extraction procedure is performed following themanufacturer's protocol.

6. MicroRNA and mRNA Isolation/Purification

Total RNAs isolated from the above Example 5 are further purified usinga mirVana™ miRNA isolation kit (Ambion, Austin, Tex.), following themanufacturer's protocol. The final products are dissolved inDEPC-treated ddH₂O and stored at −80° C. before use. Because bacterialRNAs are degraded very fast (a few hours) in nature while eukaryoticpoly-A RNAs (mRNAs) and hairpin-like microRNA precursors (pre-miRNA orpri-miRNA) remain relatively stable at 4° C. (half-life up to 3-4 days),we can use this difference to acquire pure mRNAs and/or pre-miRNAs forfurther applications. For example, RGFP mRNA can be used to identify thetransfected cells, while pre-miR-302s are used to reprogram somaticcells to ESC-like iPS cells. The purified pre-miR-302s can also be addedinto stem cell culture medium to facilitate and maintain thereprogramming process.

7. Immunostaining Assay

Embedding, sectioning and immunostaining tissue samples are performed asreported (Lin et al., RNA 2008). Primary antibodies include Oct4 (SantaCruz), Sox2 (Santa Cruz), Nanog (Santa Cruz), and RGFP (Clontech).Fluorescent dye-labeled goat anti-rabbit or horse anti-mouse antibody isused as the secondary antibody (Invitrogen-Molecular Probes). Positiveresults are examined and analyzed at 100× or 200× magnification under afluorescent 80i microscopic quantitation system with a Metamorph imagingprogram (Nikon).

8. Bisulfite DNA Sequencing

Genomic DNAs are isolated from about two million cells using a DNAisolation kit (Roche, Indianapolis, Iowa) and 1 μg of the isolated DNAsare further treated with bisulfite (CpGenome DNA modification kit,Chemicon, Temecula, Calif.), according to the manufacturers'suggestions. The treatment with bisulfite converts all unmethylatedcytosine to uracil, while methylated cytosine remains as cytosine. Forbisulfite DNA sequencing analyses, we amplify the promoter regions ofOct4 and Nanog with PCR. Primers include 5′-GAGGCTGGAG CAGAAGGATTGCTTTGG-3′(SEQ.ID.NO.2) and 5′-CCCTCCTGAC CCATCACCTCCACCACC-3′(SEQ.ID.NO.3) for Oct4, and 5′-TGGTTAGGTT GGTTTTAAAT TTTTG-3′(SEQ.ID.NO.4) and 5′-AACCCACCCT TATAAATTCT CAATTA-3′(SEQ.ID.NO.5) forNanog. The bisulfite-modified DNAs (50 ng) are first mixed with theprimers (total 100 pmole) in 1×PCR buffer, heated to 94° C. for 2 min,and immediately cooled on ice. Next, 25 cycles of PCR are performed asfollows: 94° C. for 1 min and 70° C. for 3 min, using an Expand HighFidelity PCR kit (Roche). The amplified DNA product with a correct sizeis further fractionized by 3% agarose gel electrophoresis, purified witha gel extraction filter (Qiagen), and then used in DNA sequencing. Adetailed profile of the DNA methylation sites is then generated bycomparing the unchanged cytosine in the converted DNA sequence to theunconverted one.

9. Cell Invasion Assay

Chamber inserts (12-μm pore size, Chemicon) were coated with 200 μg/mlof Matrigel alone or supplemented with 20% FBS in phenol red-free-DMEMwith 1% L-glutamine and dried overnight under sterile conditions. Cellswere harvested, washed, and resuspended in phenol red-free-DMEM to givea final cell density of 1×10⁵ cells/ml. Five hundred microliters of theresulting cell suspension was then dispensed into the top chamberwhereas DMEM conditioned medium (1.5 ml) was added to the bottom chamberto create a chemotactic gradient. Invasion was measured after overnightincubation at 37° C. for 16 hours. Top chambers were wiped with cottonwool, and invading cells on the underside of the membrane were fixed in100% methanol for 10 min, air dried, stained in cresyl violet for 20min, and gently rinsed in water. When dry, the cresyl violet stain onmembranes was eluted using a 100% ethanol/0.2 M NaCitrate (1:1) wash for20 min and absorbance read at 570 nm using a Precision Microplate Reader(Molecular Dynamics). The percentage of invading cells was calculated bycomparison of absorbance in test samples against absorbance determinedon membrane inserts that were not wiped (total cells). The result wasshown in FIG. 10A.

10. Cell Adhesion Assay

Cell Adhesion assay was performed as reported (Lin et al., Cancer Res.2010). Human bone marrow endothelial cells (hBMECs) were seeded at adensity of 1×10⁵ cells/ml in 96-well plates and washed with adhesionmedium [RPMI 1640/0.1% BSA/20 mM HEPES (pH7.4)] before assays. Testedcells were trypsinized (tumor/cancer cells) or collagenase-digested(mirPS cells), washed in sterile saline, and resuspended at 1×10⁶cells/ml in PBS with 10 μM fura-4 acetoxymethyl ester (fluorescentprobe, Sigma) for 1 hour at 37° C. in the dark. The cells were thenpelleted, washed in serum-free medium containing 1% (v/v) of probenecid(100 mM) and incubated for 20 min in adhesion medium at 37° C. in thedark to activate the intracellular fluorescent probe. After that, 10⁵cells (in 300-μl cell suspension/well) were added to the confluent hBMECendothelial monolayer and incubated for 50 min at 37° C. Non-adherentcells were removed using 2×250 μl washes of adhesion medium. Plates wereread in a fluorescent plate reader (Molecular Dynamics) at 37° C. usingan excitation wavelength of 485 nm and an emission wavelength of 530 nm.The result was shown in FIG. 10B.

11. Implantation and Teratoma Formation

Approximately 5-10 mirPS cell-derived embryoid bodies (4- to8-cell-stage) were suspended in 50 μl of a mixture of DMEM and Matrigel(2:1), followed by implantation into the uterus of a 6-week-old femalepseudopregnant immunocompromised SCID-beige mouse. The pseudopregnantmice were prepared by intraperitoneal injection of 1 IU human menopausalgonadotrophin (HMG) for two days and then human chorionic gonadotrophin(hCG) for one more day. The mice were anesthetized with 2.5% Avertinsolution, 0.4 ml per mouse during implantation. Xenografted masses weremonitored 3-4 weeks after the implantation or when the sizes were grownto over 100 mm³. Cysts/teratomas were dissected and the volumes werecalculated using the formula (length×width²)/2. Cyst/teratoma lesionswere counted, weighed and subjected to further histological analysis.Formation of teratoma-like tissue cysts was usually observed atapproximately 2.5-week post-implantation. The result was shown in FIG.11A.

12. In Vivo Tumorigenicity Assay

We xenografted Tera-2 cells (2×10⁶ cells in a total volume of 100 μlMatrigel-PBS) into the flanks (e.g. right hind limb) of eight-week-oldmale mice (BALB/c nu/nu strain). Tumors were monitored weekly and insitu injection of either pre-miR-302s or pre-miR-302-encoding plasmidvectors, e.g. pCMV-miR302s vector or pCMV-miR302d*, was conducted oneweek after the Tera-2 xenograft. Five treatments (three-day intervalsfor each treatment) of 2 μg PEI-formulated pCMV-miR302s or pCMV-miR302d*vector (total 10 μg) per g mouse weight were performed. In vivo-jetPEIDelivery Reagent (Polyplus-transfection Inc., New York, N.Y.) was usedas the manufacturer's suggestion. Samples were collected either threeweeks post injection or when untreated tumors grew to an average size ofapproximately 100 mm³. Major organs, such as the blood, brain, heartlung, liver, kidney and spleen, and the xenografts were removed forhistological evaluation of tumor lesions and immunoreactivecytotoxicity. Tumor formation was monitored by palpation and tumorvolume was calculated using the formula (length×width²)/2. Tumor lesionswere counted, dissected, weighed, and subjected to histologicalexamination using H&E and immunostaining assays. Histologicalexamination showed no detectable tissue lesions in brain, heart, lung,liver, kidney and spleen. The results were shown in FIGS. 11A and 11C.

13. In Vivo Wound Healing Test

The pre-miR-302s and their related plasmid vectors were amplified asdescribed in Examples 1 and 5 and extracted as described in Examples 5and 6. Then, the isolated pre-miR-302s and their related plasmid vectorswere mixed with a pre-prepared ointment base containing cocoa butter,cottonseed oil, olive oil, sodium pyruvate, and white petrolatum. Theconcentration of miR-302 precursors and vectors in the prepared ointmentbase is 10 μg/mL. Skin open wounds were generated by scalpel dissection;approximately 0.5-cm for control and 1.0-cm for treated mice. Ointment(about 0.3 mL) was directly applied on the wound and covered the wholewounded area. Then, the treated area was further sealed by liquidbandage.

14. Statistic Analysis

Any change over 75% of signal intensity in the analyses ofimmunostaining, western blotting and northern blotting is considered asa positive result, which in turn is analyzed and presented as mean±SE.Statistical analysis of data is performed by one-way ANOVA. When maineffects are significant, the Dunnett's post-hoc test is used to identifythe groups that differ significantly from the controls. For pairwisecomparison between two treatment groups, the two-tailed student t testis used. For experiments involving more than two treatment groups, ANOVAis performed followed by a post-hoc multiple range test. Probabilityvalues of p<0.05 is considered significant. All p values are determinedfrom two-tailed tests.

Sequence Listing:

(1) GENERAL INFORMATION:

-   -   (iii) NUMBER OF SEQUENCES: 5        (2) INFORMATION FOR SEQ ID NO:1:    -   (i) SEQUENCE CHARACTERISTICS:        -   (A) LENGTH: 23 base pairs        -   (B) TYPE: nucleic acid        -   (C) STRANDEDNESS: single        -   (D) TOPOLOGY: linear    -   (ii) MOLECULE TYPE: other nucleic acid        -   (A) DESCRIPTION: /desc=“synthetic”    -   (iii) HYPOTHETICAL: NO    -   (iv) ANTI-SENSE: YES    -   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    -   TCACTGAAAC ATGGAAGCAC TTA        (2) INFORMATION FOR SEQ ID NO:2:    -   (i) SEQUENCE CHARACTERISTICS:        -   (A) LENGTH: 27 base pairs        -   (B) TYPE: nucleic acid        -   (C) STRANDEDNESS: single        -   (D) TOPOLOGY: linear    -   (ii) MOLECULE TYPE: other nucleic acid        -   (A) DESCRIPTION: /desc=“synthetic”    -   (iii) HYPOTHETICAL: NO    -   (iv) ANTI-SENSE: NO    -   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GAGGCTGGAG CAGAAGGATT        GCTTTGG        (2) INFORMATION FOR SEQ ID NO:3:    -   (i) SEQUENCE CHARACTERISTICS:        -   (A) LENGTH: 27 base pairs        -   (B) TYPE: nucleic acid        -   (C) STRANDEDNESS: single        -   (D) TOPOLOGY: linear    -   (ii) MOLECULE TYPE: other nucleic acid        -   (A) DESCRIPTION: /desc=“synthetic”    -   (iii) HYPOTHETICAL: NO    -   (iv) ANTI-SENSE: NO    -   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CCCTCCTGAC CCATCACCTC        CACCACC        (2) INFORMATION FOR SEQ ID NO:4:    -   (i) SEQUENCE CHARACTERISTICS:        -   (A) LENGTH: 25 base pairs        -   (B) TYPE: nucleic acid        -   (C) STRANDEDNESS: single        -   (D) TOPOLOGY: linear    -   (ii) MOLECULE TYPE: other nucleic acid        -   (A) DESCRIPTION: /desc=“synthetic”    -   (iii) HYPOTHETICAL: NO    -   (iv) ANTI-SENSE: NO    -   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: TGGTTAGGTT GGTTTTAAAT        TTTTG        (2) INFORMATION FOR SEQ ID NO:5:    -   (i) SEQUENCE CHARACTERISTICS:        -   (A) LENGTH: 25 base pairs        -   (B) TYPE: nucleic acid        -   (C) STRANDEDNESS: single        -   (D) TOPOLOGY: linear    -   (ii) MOLECULE TYPE: other nucleic acid        -   (A) DESCRIPTION: /desc=“synthetic”    -   (iii) HYPOTHETICAL: NO    -   (iv) ANTI-SENSE: NO    -   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: AACCCACCCT TATAAATTCT        CAATTA

ADVANTAGES AND CLAIMS

The advantages of the present invention include: first, cost-effectiveproduction due to the fast growth of bacteria; second, easy handlingbecause of no need for culturing dedicate hybridomas or mammalian cells;third, high product quality in view of the pol-2 promoter-liketranscription and improved reading fidelity of the prokaryotictranscription machinery; fourth, industrial level bulk production fordesired RNAs and their related peptides/proteins as well as theintroduced plasmid vectors all at once in the bacteria; and last,multiple task capacity in that the desired RNAs and proteins can beseparately isolated and purified from the bacterial extracts and/orlysates for further applications. Therefore, taken together, aninducible method for producing RNAs (i.e. messenger RNAs and microRNAs)and/or peptides/proteins (i.e. antibodies, growth factors and enzymes)using eukaryotic RNA promoter-driven transcription in prokaryotic cellsis highly desirable.

It will be apparent to those skilled in the art that variousmodification and variations can be made in the method and relatedapparatus of the present invention without departing from the spirit orscope of the invention. Thus, it is intended that the present inventioncover modifications and variations that come within the scope of theappended claims and their equivalents.

What is claimed is:
 1. A composition for regulating eukaryoticelongation factor-1 alpha (EF1alpha) promoter-driven gene expression inprokaryotes, comprising: (a) at least a chemical agent capable ofinducing or enhancing eukaryotic EF1alpha promoter-driven geneexpression in prokaryotic cells, wherein said chemical agent containinga structure of 3-morpholinopropane-1-sulfonic acid (MOPS); and (b) aplurality of prokaryotic cells, said plurality of prokaryotic cellscontaining at least a gene mediated by a eukaryotic EF1alphapromoter-driven expression mechanism and wherein said gene encodes atleast a hairpin-like structure in its 5′-untranslated region (5′-UTR)for being expressed in prokaryotes; wherein (a) and (b) are mixedtogether under a condition to induce the eukaryotic EF1alphapromoter-driven gene expression of said gene in said prokaryotic cells;wherein said condition is in Luria-Bertani (LB) broth.
 2. Thecomposition as defined in claim 1, wherein each one of said prokaryoticcells has at least an inducible gene expression composition.
 3. Thecomposition as defined in claim 2, wherein said gene is located in aplasmid vector containing at least a eukaryotic EF1alpha promoter. 4.The composition as defined in claim 1, wherein said gene encodes atleast a non-coding RNA and a protein-coding RNA.
 5. The composition asdefined in claim 4, wherein said non-coding RNA is a small hairpin RNA.6. The composition as defined in claim 4, wherein said protein-codingRNA further encodes a red fluorescent protein (RGFP).
 7. The compositionas defined in claim 4, wherein said protein-coding RNA contains 30% to100% homology to a cDNA sequence of a eukaryotic gene.
 8. Thecomposition as defined in claim 4, wherein said non-coding RNA or saidprotein-coding RNA is isolatable from said prokaryotes.
 9. Thecomposition as defined in claim 1, wherein said gene encodes at least aprotein.
 10. The composition as defined in claim 4, wherein a proteintranscribed from said protein-coding RNA contains at least a peptidesequence.
 11. The composition as defined in claim 10, wherein saidprotein or said peptide is isolatable from said prokaryotes.
 12. Thecomposition as defined in claim 4, wherein a protein transcribed fromsaid protein-coding RNA is an enzyme.
 13. The composition as defined inclaim 1, wherein said prokaryotic cells are bacterial cells.
 14. Thecomposition as defined in claim 1, wherein said prokaryotic cells areEscherichia coli (E. coli).
 15. The composition as defined in claim 1,wherein said chemical agent is a transcription inducer for inducing RNAexpression of said gene encoding at least the hairpin-like structure insaid prokaryotic cells.
 16. The composition as defined in claim 1,wherein said chemical agent in said condition has a v/v concentration of0.01% to 1%.
 17. The composition as defined in claim 4, wherein saidnon-coding RNA is useful for pharmaceutical or therapeutic application.18. The composition as defined in claim 4, wherein said protein-codingRNA is useful for pharmaceutical or therapeutic application.
 19. Thecomposition as defined in claim 4, wherein said non-coding RNA is aprecursor of microRNA.
 20. The composition as defined in claim 19,wherein said precursor of microRNA is a precursor of miR-302.
 21. Thecomposition as defined in claim 4, wherein a protein transcribed fromsaid protein-coding RNA is useful for pharmaceutical or therapeuticapplication.
 22. The composition as defined in claim 17, wherein saidpharmaceutical or therapeutic application is cancer therapy and diseasetreatment.